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MED12, the Mediator Complex Subunit 12 Gene, Is Mutated at High Frequency in Uterine Leiomyomas

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Science  14 Oct 2011:
Vol. 334, Issue 6053, pp. 252-255
DOI: 10.1126/science.1208930

Abstract

Uterine leiomyomas, or fibroids, are benign tumors that affect millions of women worldwide and that can cause considerable morbidity. To study the genetic basis of this tumor type, we examined 18 uterine leiomyomas derived from 17 different patients by exome sequencing and identified tumor-specific mutations in the mediator complex subunit 12 (MED12) gene in 10. Through analysis of 207 additional tumors, we determined that MED12 is altered in 70% (159 of 225) of tumors from a total of 80 patients. The Mediator complex is a 26-subunit transcriptional regulator that bridges DNA regulatory sequences to the RNA polymerase II initiation complex. All mutations resided in exon 2, suggesting that aberrant function of this region of MED12 contributes to tumorigenesis.

Uterine leiomyomas, also called fibroids, are benign tumors that occur in 60% of women by the age of 45 years and that cause symptoms in about half of the cases (1). These symptoms include abdominal pain and discomfort and abnormal bleeding. Uterine leiomyomas are also an important cause of infertility [reviewed in (2, 3)], and they are the most common medical reason for hysterectomy (4). Several recurrent genetic aberrations such as deletions in 7q, trisomy of chromosome 12, and various rearrangements affecting the high mobility group AT-hook 2 (HMGA2) gene mapping to chromosome 12q14 (57) have been observed in uterine leiomyomas, but these occur at low frequency. To investigate whether these tumors have high-frequency genetic alterations, we investigated all protein-coding genes by exome sequencing in 18 uterine leiomyomas and the respective normal tissue DNAs. These tumors came from 17 different patients.

The most frequent tumor-specific alterations in the set of 18 tumors affected the MED12 gene on chromosome Xq13.1. MED12 is a subunit of the Mediator complex, which is thought to regulate global, as well as gene-specific, transcription (8). Ten tumors displayed a mutation, and eight of these were located in codon 44 (Transcript ENST00000374080) (P = 1.3 × 10−36). Furthermore, one mutation was in the neighboring codon 43, and another deleted the 3 base pairs (bp) encoding codon 36. No mutations in the remaining 44 MED12 exons were observed.

To validate the finding, we examined the original set of 18 and an additional set of 207 uterine leiomyomas for codon 44 and flanking sequence mutations by capillary sequencing. In total, 225 tumors derived from 80 different individuals were evaluated. This analysis confirmed a notable frequency of somatic MED12 codon 44 region mutations in uterine leiomyomas (Table 1 and fig. S2). Of the 225 tumors, 110 (49%) displayed missense mutations affecting codon 44. These included G44V, G44C, G44R, G44A, G44S, and G44D mutations (Figs. 1 and 2). Q43P was found three times. Another hot spot was detected in codon 36. Eleven lesions (5%) all displayed a L36R mutation. Twenty-five tumors (11%) with an insertion-deletion type mutation were also observed (Table 1 and Fig. 2). All of these are predicted to result in an in-frame transcript. Detailed results for each patient and each tumor are shown in table S5.

Table 1

Summary of somatic MED12 mutations observed in the series of 225 unselected uterine leiomyomas.

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Fig. 1

Sequence chromatograms showing somatic mutations in MED12 codon 44 in human uterine leiomyomas. Examples of genomic DNA and cDNA sequencing traces in codon 44 mutated samples and a wild-type sample are shown. Codon 44 is highlighted by the horizontal bars above the traces. Mutated bases are indicated by arrows.

Fig. 2

Detailed view of MED12 exon 2. (Top) Exon 2 of MED12 and the observed deletions and insertions in the sequenced leiomyomas. Amino acid translation and codon number are shown below the DNA sequence. (Bottom) Multispecies alignment of the mutation hot spot near the beginning of exon 2. Arrows indicate observed missense and intronic mutations at each base, and numbers above the arrows indicate how many times each mutation was detected in this study of 225 tumors. Amino acid numbering and secondary structure predictions produced by protein structure prediction server PSIpred are shown below the alignment (C, coil; H, helix).

A somatic intronic T to A mutation was identified 8 bp upstream of the splice acceptor site of exon 2 in 10 tumors (4%). This change was predicted to create an aberrant splice acceptor site in intron 1 and to extend the transcript by the last six bases of intron 1. In three of these cases, RNA was available for cDNA sequencing, and in all three cases we confirmed the addition of the predicted six bases to the transcript. Thus, in total, 159 (70%) of the 225 uterine leiomyomas displayed a mutation affecting MED12 exon 2. Of the 80 individual patients, 60 (75%) had tumor(s) with MED12 mutations. The MED12 mutations in the tumors were heterozygous, and no tumor displayed more than one mutation (P = 2.2 × 10−28). Finally, 20 mutation-negative and 10 mutation-positive samples were analyzed for all 45 MED12 exons by Sanger sequencing (9). This analysis revealed no additional MED12 mutations.

We next investigated whether MED12 mutation status correlated with patient age at hysterectomy or with tumor size. No correlation was detected between the MED12 mutation status and patient age at hysterectomy (P = 0.69), but we found that tumors lacking MED12 mutations tended to be larger (P = 0.015). The 66 tumors lacking MED12 mutations were not randomly distributed among the studied patients but aggregated within a subset of them (P < 10−6) (tables S2, S3, and S5).

X-chromosomal genes in normal cells typically display monoallelic expression in females due to random inactivation of one of the two X chromosomes (10). This phenomenon has been used extensively in studies on clonality; in multicellular lesions originating from a single cell, the active X is typically the same in all cells (11).To confirm that the MED12 mutant alleles are expressed in the tumors, we isolated and sequenced cDNA from a total of 16 tumors from 16 patients. Of these, 14 had codon 44 mutations, one had a codon 43 mutation, and one had a codon 36 mutation. In each case, predominantly the mutant allele was present in the cDNA sequence (Fig. 1) (P = 1.5 × 10−5).

The Mediator complex consists of 26 subunits and is thought to regulate transcription by bridging regulatory elements such as enhancers to the RNA polymerase II initiation complex [reviewed in (8)]. The Mediator complex is highly conserved in all eukaryotes and is required for the transcription of almost all genes in yeast (12). Together with MED13, CDK8, and Cyclin C, MED12 forms a Mediator subcomplex known as a kinase or CDK8 module (1315). This complex is suggested to have a role in transcriptional repression (16, 17), but it also seems to act as a positive coregulator (e.g., of p53 target genes) (18). MED12 also directly interacts with β-catenin and is required for the cellular response to Wnt signals (19). Reduced expression of direct Wnt/β-catenin signaling target genes cyclin D1, Axin2, and Myc has been shown in Med12 hypomorphic mouse embryos (20). MED12 has also been shown to modulate the sonic hedgehog signaling pathway through Gli3 interaction (21). The Mediator complex is believed to have roles in general as well as gene-specific transcription (8). We speculate that the tumorigenic effect of the mutations in uterine smooth muscle, but not in many other tissue types, may relate to the latter.

To determine whether the tumor-associated mutations in MED12 correlate with alterations in global gene expression patterns in the tumors, we examined the Affymetrix Human Genome U133 Plus 2.0 gene expression array data that were available for 10 tumors and 10 matched normal myometrium specimens. Four tumors displayed a codon 44 mutation, four carried the intron 1 mutation, and the remaining two displayed no MED12 mutation. The gene expression data from the mutation-positive lesions were compared to those of mutation-negative lesions as well as normal myometrium specimens in unsupervised hierarchical clustering (9). The MED12 mutation-positive specimens clustered separately from the other specimens (fig. S1). Although interesting, this correlation could reflect changes other than the MED12 mutations; thus, the finding needs to be validated in future studies with a larger number of samples.

We also performed pathway analysis, comparing eight tumors positive for MED12 mutations with their respective normal tissues. After multiple testing correction, three pathways were found to be substantially altered in the tumors—namely, the pathways for focal adhesion, extracellular matrix receptor interaction, and Wnt signaling (9).

The genetic evidence suggests that MED12 mutations contribute to the genesis of uterine leiomyomas. All the observed mutations affect an evolutionarily conserved region of the MED12 protein. Furthermore, the glycine at codon 44 is the most conserved of these key residues, being conserved across all 39 species examined, including plants and fungi (22). This codon (Fig. 2) appears to be indispensable for normal MED12 function in uterine myometrium because our set of tumors displayed all six possible base substitutions leading to the change of this residue (Fig. 1).

The mutant transcript appears to be relevant for tumorigenesis because the aberrant transcript was predominant as compared to the wild-type transcript in all 16 tumor sample-derived cDNAs examined (Fig. 1) (P = 1.5 × 10−5). Although expression of normal transcript from a subclone within the tumor cannot be excluded, a plausible explanation for the minor normal transcript peak visible in the analyses is normal tissue contamination.

Germline MED12 exon 21 mutations R961W and G958E underlie Opitz-Kaveggia syndrome (MIM#305450), a rare condition with congenital anomalies and mental retardation (23, 24). Mutation N1007S has been implicated in Lujan-Fryns syndrome (MIM#309520), characterized by mental retardation and syndromic features such as unusual height, long limbs, and long, thin fingers (25). These syndromes are not associated with tumor predisposition, and the localization of the leiomyoma mutations within the MED12 gene is strikingly different from that in the other disorders, suggesting that the molecular mechanisms underlying the conditions are likely to be different.

In summary, the discovery of frequent MED12 mutations in uterine leiomyomas should fuel further work to elucidate the role of this protein, and the Mediator complex in general, in both normal cell biology and tumorigenesis.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1208930/DC1

Materials and Methods

Figs. S1 and S2

Tables S1 to S7

References (2629)

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Acknowledgments: We thank S. Nieminen, I.-L. Svedberg, M. Kuris, and I. Vuoristo for technical assistance. This work was supported by grants from the Academy of Finland (Center of Excellence in Translational Genome-Scale Biology grant 6302352, and grants 1124270, 212901, and 214323), European Commission Projects TCAC in Cancer (project number 2005-518200) and GENICA Genomic Instability in Cancer and Precancer (project number 201630), Sigrid Jusélius Foundation, and the Cancer Society of Finland. Primer sequences are provided in the Supporting Online Material. The gene expression data presented have been deposited in the Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo) and are accessible with the accession number GSE30673.
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